This project's goal is to determine the protein structures, dynamics, and interactions that govern the active transport of calcium in muscle, to pave the way for rational therapeutic design in muscle disease. We focus on two integral membrane proteins, the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) and its principal regulator in the heart, phospholamban (PLB). Our core technology is site-directed spectroscopy in both purified proteins and living cells. We will develop and apply innovative and complementary methods in site-directed labeling, fluorescence, and EPR, combined with NMR and crystallographic data obtained from our collaborators. All of this will be integrated and informed by computational simulations of both spectroscopic and molecular dynamics to analyze protein structure, dynamics, and interactions. Aims 1 and 2 deal with applications to fundamental mechanisms in this system, and Aims 3 and 4 combine these techniques and fundamental insights to move toward biophysically designed therapies. In Aim1, we investigate the fundamental functional dynamics of SERCA, to determine the key transitions in structure and motion that govern its catalytic mechanism, {focusing on steps that are crucial for substrate activation and regulation}. Aim2 investigates the mechanism by which PLB regulates SERCA, adding themes of protein-protein interaction and phosphorylation. Aim3 uses the insights of the first two aims, based primarily on discoveries of the previous project period, to design PLB mutants that are strong candidates for future gene therapy applications. Similarly, Aim4 uses live-cell fluorescence assays of SERCA and PLB, in conjunction with our new technology in high-throughput fluorescence lifetime measurement, to develop and apply novel small- molecule screens, with the ultimate goal of drug discovery for treatment of heart failure and other disorders of calcium homeostasis. {The four Aims are synergistic, strengthening each other with new discoveries and hypotheses to be tested, but they are not interdependent, since feasibility has been established independently for each aim and subaim.} This research brings together a powerful combination of techniques, concepts, and collaborators, from biophysics to chemical biology to molecular genetics, to solve the molecular mechanisms of calcium transport and regulation in muscle. This project remains grounded in fundamental biophysical mechanisms, but it has recently become clear that SERCA is one of the most important therapeutic targets for some of the greatest health problems, including heart failure, muscular dystrophy, diabetes, and cancer. It is also clear that the biophysical tools being developed in this project have matured to the point where they can play a crucial role not only in understanding the functions of SERCA and PLB, but also in controlling these functions. Therefore, the collaborative team now includes scientists with strong track records in drug development and gene therapy. It is anticipated that by the end of the next funding period, this project will stimulate separate efforts in truly translational research on muscle therapeutics.